• Biological and Clinical Factors Contributing to the Metabolic Heterogeneity of Hospitalized Patients with and without COVID-19

      D'Alessandro, Angelo; Thomas, Tiffany; Akpan, Imo J; Reisz, Julie A; Cendali, Francesca I; Gamboni, Fabia; Nemkov, Travis; Thangaraju, Kiruphagaran; Katneni, Upendra; Tanaka, Kenichi; et al. (MDPI AG, 2021-09-02)
      The Corona Virus Disease 2019 (COVID-19) pandemic represents an ongoing worldwide challenge. The present large study sought to understand independent and overlapping metabolic features of samples from acutely ill patients (n = 831) that tested positive (n = 543) or negative (n = 288) for COVID-19. High-throughput metabolomics analyses were complemented with antigen and enzymatic activity assays on plasma from acutely ill patients collected while in the emergency department, at admission, or during hospitalization. Lipidomics analyses were also performed on COVID-19-positive or -negative subjects with the lowest and highest body mass index (n = 60/group). Significant changes in amino acid and fatty acid/acylcarnitine metabolism emerged as highly relevant markers of disease severity, progression, and prognosis as a function of biological and clinical variables in these patients. Further, machine learning models were trained by entering all metabolomics and clinical data from half of the COVID-19 patient cohort and then tested on the other half, yielding ~78% prediction accuracy. Finally, the extensive amount of information accumulated in this large, prospective, observational study provides a foundation for mechanistic follow-up studies and data sharing opportunities, which will advance our understanding of the characteristics of the plasma metabolism in COVID-19 and other acute critical illnesses.
    • Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage.

      Kim, Christopher Y; Johnson, Hannah; Peltier, Sandy; Spitalnik, Steven L; Hod, Eldad A; Francis, Richard O; Hudson, Krystalyn E; Stone, Elizabeth F; Gordy, Dominique E; Fu, Xiaoyun; et al. (Frontiers Media S.A., 2022-04-26)
      Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality.
    • Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients

      Thomas, Tiffany; Stefanoni, Davide; Dzieciatkowska, Monika; Issaian, Aaron; Nemkov, Travis; Hill, Ryan C; Francis, Richard O; Hudson, Krystalyn E; Buehler, Paul W; Zimring, James C; et al. (American Chemical Society, 2020-10-26)
      The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.
    • The Impact of Age and BMI on the VWF/ADAMTS13 Axis and Simultaneous Thrombin and Plasmin Generation in Hospitalized COVID-19 Patients.

      Thangaraju, Kiruphagaran; Katneni, Upendra; Akpan, Imo J; Tanaka, Kenichi; Thomas, Tiffany; Setua, Saini; Reisz, Julie A; Cendali, Francesca; Gamboni, Fabia; Nemkov, Travis; et al. (Frontiers Media S.A., 2022-01-10)
      Aging and obesity independently contribute toward an endothelial dysfunction that results in an imbalanced VWF to ADAMTS13 ratio. In addition, plasma thrombin and plasmin generation are elevated and reduced, respectively, with increasing age and also with increasing body mass index (BMI). The severity risk of Corona Virus Disease 2019 (COVID-19) increases in adults older than 65 and in individuals with certain pre-existing health conditions, including obesity (>30 kg/m2). The present cross-sectional study focused on an analysis of the VWF/ADAMTS13 axis, including measurements of von Willebrand factor (VWF) antigen (VWF:AG), VWF collagen binding activity (VWF:CBA), Factor VIII antigen, ADAMTS13 antigen, and ADAMTS13 activity, in addition to thrombin and plasmin generation potential, in a demographically diverse population of COVID-19 negative (-) (n = 288) and COVID-19 positive (+) (n = 543) patient plasmas collected at the time of hospital presentation. Data were analyzed as a whole, and then after dividing patients by age (<65 and ≥65) and independently by BMI [<18.5, 18.5-24.9, 25-29.9, >30 (kg/m2)]. These analyses suggest that VWF parameters (i.e., the VWF/ADAMTS13 activity ratio) and thrombin and plasmin generation differed in COVID-19 (+), as compared to COVID-19 (-) patient plasma. Further, age (≥65) more than BMI contributed to aberrant plasma indicators of endothelial coagulopathy. Based on these findings, evaluating both the VWF/ADAMTS13 axis, along with thrombin and plasmin generation, could provide insight into the extent of endothelial dysfunction as well as the plasmatic imbalance in coagulation and fibrinolysis potential, particularly for at-risk patient populations.
    • The interactome of the N-terminus of band 3 regulates red blood cell metabolism and storage quality

      Issaian, Aaron; Hay, Ariel; Dzieciatkowska, Monika; Roberti, Domenico; Perrotta, Silverio; Darula, Zsuzsanna; Redzic, Jasmina; Busch, Micheal P; Page, Grier P; Rogers, Stephen C; et al. (Ferrata-Storti Foundation, 2021-05-13)
      Band 3 (anion exchanger 1 - AE1) is the most abundant membrane protein in red blood cells (RBCs), the most abundant cell in the human body. A compelling model posits that - at high oxygen saturation - the N-term cytosolic domain of AE1 binds to and inhibits glycolytic enzymes, thus diverting metabolic fluxes to the pentose phosphate pathway to generate reducing equivalents. Dysfunction of this mechanism occurs during RBC aging or storage under blood bank conditions, suggesting a role for AE1 in the regulation of blood storage quality and efficacy of transfusion - a life-saving intervention for millions of recipients worldwide. Here we leverage two murine models carrying genetic ablations of AE1 to provide mechanistic evidence of its role in the regulation of erythrocyte metabolism and storage quality. Metabolic observations in mice recapitulated those in a human subject lacking expression of AE11-11 (band 3 Neapolis), while common polymorphisms in the region coding for AE11-56 correlate with increased susceptibility to osmotic hemolysis in healthy blood donors. Through thermal proteome profiling and cross-linking proteomics, we provide a map of the RBC interactome, with a focus on AE11-56 and validate recombinant AE1 interactions with glyceraldehyde 3-phosphate dehydrogenase (GAPDH). As a proof-of-principle and further mechanistic evidence of the role of AE1 in the regulation of redox homeostasis of stored RBCs, we show that incubation with a cell-penetrating AE11-56 peptide can rescue the metabolic defect in glutathione recycling and boost post-transfusion recoveries of stored RBCs from healthy human donors and genetically ablated mice.
    • ZOOMICS: Comparative Metabolomics of Red Blood Cells From Guinea Pigs, Humans, and Non-human Primates During Refrigerated Storage for Up to 42 Days.

      Bertolone, Lorenzo; Shin, Hye Kyung H; Baek, Jin Hyen; Gao, Yamei; Spitalnik, Steven L; Buehler, Paul W; D'Alessandro, Angelo (Frontiers Media S.A., 2022-03-21)
      Unlike other rodents, guinea pigs (Cavia porcellus) have evolutionarily lost their capacity to synthesize vitamin C (ascorbate) de novo and, like several non-human primates and humans, rely on dietary intake and glutathione-dependent recycling to cope with oxidant stress. This is particularly relevant in red blood cell physiology, and especially when modeling blood storage, which exacerbates erythrocyte oxidant stress. Herein we provide a comprehensive metabolomics analysis of fresh and stored guinea pig red blood cell concentrates (n = 20), with weekly sampling from storage day 0 through 42. Results were compared to previously published ZOOMICS studies on red blood cells from three additional species with genetic loss of L-gulonolactone oxidase function, including humans (n = 21), olive baboons (n = 20), and rhesus macaques (n = 20). While metabolic trends were comparable across all species, guinea pig red blood cells demonstrated accelerated alterations of the metabolic markers of the storage lesion that are consistent with oxidative stress. Compared to the other species, guinea pig red blood cells showed aberrant glycolysis, pentose phosphate pathway end product metabolites, purine breakdown products, methylation, glutaminolysis, and markers of membrane lipid remodeling. Consistently, guinea pig red blood cells demonstrated higher end storage hemolysis, and scanning electron microscopy confirmed a higher degree of morphological alterations of their red blood cells, as compared to the other species. Despite a genetic inability to produce ascorbate that is common to the species evaluated, guinea pig red blood cells demonstrate accelerated oxidant stress under standard storage conditions. These data may offer relevant insights into the basal and cold storage metabolism of red blood cells from species that cannot synthesize endogenous ascorbate.